ABSTRACT Otitis media (OM) is the most frequently diagnosed illness in children and a leading cause of hearing loss. OM and other diseases of the upper and lower respiratory tract caused by NTHI continue to be a significant health and socioeconomic problem in both children and adults. Despite this, we do not yet understand the dynamics of NTHI survival in the host, both as a commensal of the human nasopharynx and opportunistic pathogen of privileged anatomical sites (i.e. middle ear). Host sequestration of essential nutrients (termed nutritional immunity) serves to control bacterial growth, particularly in privileged sites. However, pathogens must overcome these barriers to growth to thrive and persist, ultimately causing disease. Our central hypothesis is that fluctuations in heme-iron availability influence the progression and severity of disease resulting in a microenvironment that potentiates NTHI persistence. Our long-term goal is to define therapeutic targets that will attenuate NTHI persistence, reduce tissue damage and diminish chronic sequelae. Here, our objective is to explore the consequence of fluctuations in essential heme-iron availability on NTHI physiology, biofilm development, invasion and disease severity. In our preliminary and published data we demonstrate that the severity of disease is directly related to the heme-iron status of NTHI at the time of introduction into the middle ear. We will test our central hypothesis through the following specific aims: 1) Determine the contribution of filamentation and DNA methylation on the biofilm developmental changes associated with fluctuations in heme-iron availability 2) Determine the contribution of heme-iron limitation on the duration and severity of OM sequelae (i.e. subclinical, symptomatic) and 3) Define the subcellular trafficking patterns of NTHI within middle ear epithelium as a consequence of heme-iron availability. We will further expand upon our novel models of symptomatic and subclinical OM to advance our understanding of the different manifestations of disease. Our investigations will increase our understanding of how NTHI adapts to nutritional limitation in the host, the influence of this adaptation on biofilm development and consequence on disease progression. In addition, the studies proposed will directly evaluate the potential utility of heme-iron sequestration through the use of agents to compete for iron on NTHI persistence to reduce the burden of NTHI-mediated OM. A better understanding of individual cell and NTHI community dynamics in the host and the microenvironmental changes that affect NTHI pathogenic lifestyles can be harnessed as potential novel therapeutics designed to ultimately thwart infectious disease states and thus significantly reduce the potential for recurrent infections and tissue damage that lead to adverse disease sequelae including hearing loss.